Method for detecting cytosine methylations

ABSTRACT

A method is described for the detection of 5-methylcytosine in genomic DNA samples. First, a genomic DNA from a DNA sample is chemically converted with a reagent, whereby 5-methylcytosine and cytosine react differently. Then the pretreated DNA is amplified with the use of a polymerase with primers of different sequence. In the next step, the amplified genomic DNA is hybridized to an oligonucleotide array and PCR products are obtained, which must be provided with a label. Alternatively, the PCR products can be extended in a primer extension reaction, wherein the extension products are also provided with a label. In the last step, the extended oligonucleotides are investigated for the presence of the label.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of U.S. patent application Ser.No. 10/311,661, filed Dec. 18, 2002, now U.S. Pat. No. 7,118,868, issuedOct. 10, 2006, which is incorporated herein by reference and which is aNational Stage of PCT/DE01/02274, filed Jun. 19, 2001, which claimspriority to German Application No. 100 29 915.6, filed Jun. 19, 2000.

BACKGROUND OF THE INVENTION

The present invention concerns a method for the detection of cytosinemethylations in DNA.

The levels of observation that have been well studied due to methoddevelopments in recent years in molecular biology include the genesthemselves, as well as [transcription and] translation of these genesinto RNA and the proteins arising therefrom. During the course ofdevelopment of an individual, when a gene is turned on and how theactivation and inhibition of certain genes in certain cells and tissuesare controlled can be correlated with the extent and nature of themethylation of the genes or of the genome. Pathogenic states are alsoexpressed by a modified methylation pattern of individual genes or ofthe genome.

5-Methylcytosine is the most frequent covalently modified base in theDNA of eukaryotic cells. For example, it plays a role in the regulationof transcription, in genetic imprinting and in tumorigenesis. Theidentification of 5-methylcytosine as a component of genetic informationis thus of considerable interest. 5-Methylcytosine positions, however,cannot be identified by sequencing, since 5-methylcytosine has the samebase-pairing behavior as cytosine. In addition, in the case of a PCRamplification, the epigenetic information, which is borne by5-methylcytosines, is completely lost.

A relatively new method that has since been applied most frequently forinvestigating DNA for 5-methylcytosine is based on the specific reactionof bisulfite with cytosine, which is converted to uracil, whichcorresponds in its base-pairing behavior to thymidine, after asubsequent alkaline hydrolysis. In contrast, 5-methylcytosine is notmodified under these conditions. Thus the original DNA is converted,such that methylcytosine, which originally cannot be distinguished fromcytosine by means of its hybridization behavior, now can be detected by“standard” molecular biological techniques as the single remainingcytosine, for example, by amplification and hybridization or sequencing.All of these techniques are based on base pairing, which now is fullyutilized. The prior art, which concerns sensitivity, is defined by amethod that incorporates the DNA to be investigated in an agarosematrix, through which diffusion and renaturation of the DNA is prevented(bisulfite reacts only on single-stranded DNA) and all precipitation andpurification steps are replaced by rapid dialysis (Olek A., et al.,Nucl. Acids Res. 1996, 24, 5064-5066). Individual cells can beinvestigated with this method, which illustrates the potential of themethod. Of course, previously, only individual regions of up toapproximately 3000 base pairs in length have been investigated; a globalinvestigation of cells for thousands of possible methylation analyses isnot possible. Of course, this method also cannot reliably analyze verysmall fragments comprised of small sample quantities. These are lostdespite the protection from diffusion through the matrix.

A review of the other known possibilities for detecting5-methylcytosines can be derived from the following review article: ReinT., DePamphilis, M. L., Zorbas H., Nucleic Acids Res. 1998, 26, 2255.

The bisulfite technique has previously been applied only in research,with a few exceptions (e.g., Ze[s]chnigk M. et al., Eur. J Hum. Gen.1997, 5, 94-8). However, short, specific pieces of a known gene arealways amplified after a bisulfite treatment and either completelysequenced (Olek A. and Walter, J., Nat. Genet. 1997, 17, 275-276) orindividual cytosine positions (Gonzalgo M. L. and Jones P. A., Nucl.Acids Res. 1997, 25, 2529-2531, WO Patent 95 00669) or an enzymecleavage (Xiong Z. and Laird P. W., Nucl. Acids Res. 1997, 25,2532-2534) are detected by a “primer extension reaction”. Also,detection by means of hybridizing has been described (Olek et al., WO99/28498).

Other publications, which are concerned with the application of thebisulfite technique for the detection of methylation in individual genesare: Xiong, Z. and Laird, P. W. (1997), Nucl. Acids Res. 25, 2532;Gonzalgo, M. L. and Jones, P. A. (1997) Nucl. Acids Res. 25, 2529; GriggS. and Clark S. (1994) Bioassays 16, 431; Zeschnigk, M. et al, (1997)Human Molecular Genetics 6, 387; Teil R. et al. (1994), Nucl. Acids Res.22, 695; Martin V. et al. (1995), Gene. 157, 261; WO 97/46705, WO95/15373 and WO 45560.

A review of the prior art in oligomer array production can be taken froma special publication of Nature Genetics that appeared in January 1999(Nature Genetics Supplement, Volume 21, January 1999), the literaturecited therein and U.S. Pat. No. 5,994,065 on methods for the productionof solid carriers for target molecules such as oligonucleotides withreduced nonspecific background signal.

Probes with many fluorescent labels have been used for the scanning ofan immobilized DNA array. Particularly suitable for fluorescent labelsis the simple introduction of Cy3 and Cy5 dyes at the 5′-OH of therespective probe. The fluorescence of the hybridized probes is detected,for example, by means of a confocal microscope. The dyes Cy3 and Cy5, inaddition to many others, are commercially available.

Matrix-assisted laser desorption/ionization mass spectrometry(MALDI-TOF) is a very powerful development for the analysis ofbiomolecules (Karas M., and Hillenkamp F. (1988) Laser desorptionionization of proteins with molecular masses exceeding 10,000 daltons.(1998) Anal. Chem. 60, 2299-2301). An analyte is embedded in alight-absorbing matrix. The matrix is evaporated by means of a shortlaser pulse and the analyte molecule is transported unfragmented intothe gas phase. The ionization of the analyte is achieved by collisionswith matrix molecules. An applied voltage accelerates the ions in afield-free flight tube. The ions are accelerated to a varying extentbased on their different masses. Smaller ions reach the detector soonerthan larger ions.

MALDI-TOF spectroscopy is excellently suitable for the analysis ofpeptides and proteins. The analysis of nucleic acids is somewhat moredifficult (Gut, I. G. and Beck, S. (1995)), DNA and Matrix AssistedLaser Desorption Ionization Mass Spectrometry. Molecular Biology:Current Innovations and Future Trends 1: 147-157.) For nucleic acids,the sensitivity is approximately 100 times poorer than for peptides anddecreases overproportionally with increasing fragment size. For nucleicacids, which have a backbone with multiple negative charges, theionization process through the matrix is essentially less efficient. InMALDI-TOF spectroscopy, the selection of the matrix plays a veryimportant role. For the desorption of peptides, several very powerfulmatrices have been found, which produce a very fine crystallization.Several high-performing matrices have been found in the meantime forDNA, but the difference in sensitivity has not been reduced in this way.The difference in sensitivity can be reduced by modifying the DNAchemically in such a way that it is similar to a peptide.

Phosphorothioate nucleic acids, in which the usual phosphates of thebackbone are substituted by thiophosphates, can be converted into acharge-neutral DNA by simple alkylation chemistry (Gut, I. G. and Beck,S. (1995), A procedure for selective DNA alkylation and detection bymass spectrometry. Nucleic Acids Res. 23: 1367-1373). The coupling of a“charge tag” to this modified DNA results in an increase in sensitivityby the same amount that is found for peptides. Another advantage of“charge tagging” is the increased stability of the analysis againstimpurities, which greatly interfere with the detection of unmodifiedsubstrates.

Genomic DNA is obtained by standard methods from DNA of cells, tissue orother test samples. This standard methodology is found in referencessuch as Fritsch and Maniatis, eds., Molecular Cloning: A LaboratoryManual, 1989.

Urea improves the efficiency of the bisulfite treatment prior tosequencing of 5-methylcytosine in genomic DNA (Paulin, R. Grigg G W,Davey M W, Piper M. (1998), Nucleic Acids Res. 26: 5009-5010).

SUMMARY OF THE INVENTION

The object of the present invention is thus to make available a methodfor the detection of cytosine methylations in DNA, which overcomes thedisadvantages of the prior art.

The object is solved in that a method for the detection of cytosinemethylations in DNA is made available, wherein the following operatingsteps are conducted:

a) a genomic DNA sample is incubated with a solution of a bisulfite(=hydrogen sulfite, disulfite) in the concentration range between 0.1and 6 mol/liter, whereby a denaturing reagent and/or solvent as well asat least one radical trap is present;

b) the treated DNA sample is diluted with water or an aqueous solution;

c) the DNA sample is amplified in a polymerase reaction;

d) one detects how much the sequence has changed by the treatmentaccording to step a) in comparison to the genomic DNA sample andconcludes the methylation state of at least one locus in the genomic DNAsample.

It is preferred according to the invention that the denaturing reagentand/or solvent is selected from the following list of compounds orcompound classes:

Polyethylene glycol dialkyl ethers, dioxane and substituted derivatives,urea or derivatives, acetonitrile, primary alcohols, secondary alcohols,tertiary alcohols, diethylene glycol dialkyl ethers, triethylene glycoldialkyl ethers, tetraethylene glycol dialkyl ethers, pentaethyleneglycol dialkyl ethers, hexaethylene glycol dialkyl ethers, DMSO, THF.

It is additionally preferred that the radical trap is selected from thefollowing group of compounds: di- and trihyroxybenzenes, green teaextract, pine bark extract, gingko biloba extract, (EGb 761), flavonoidmixture of different fruit and vegetable extracts (GMLD), Bio-Normalizer(Sun-O Corp), DPPH (1,1-diphenyl-2-picrylhydrazyl), NDGA(nordihydroguaiaretic acid), Trolox(6-hydroxy-2,5-7,8-tetramethylchromane 2-carboxylic acid),2,6-di-tert-butylphenol, 4-methyl-di-tert-butylphenol,4-methoxy-di-tert-butylphenol, 2,6-di-tert-butyl-p-cresol,3,4-dihydroxybenzoic acid, vitamin C, vitamin E, vitamin Q,hydroquinone, ubiquinone, lignans, hydroxyterpenes, flavonoids,curcumin, tannins, retinoic acid compounds, Ge-132bis-beta-carboxyethylgermanium sesquioxide, superoxide dismutase (SOD),superoxide catylase, alpha-naphthoflavone,di(2-methyl-5-chlorophenyl)dithionate and Cu(II) derivatives,mebendazole, CS (chloroform-soluble) alkyloid extract,4-(3,5-di-tert-butyl-4-hydroxyphenyl) 3-hydroxy-1,2-naphthoquinone,4-(3,5-di-tert-butyl-4-hydroxyphenyl) 3-methoxy-1,2-naphthoquinone,4-(3,5-di-tert-butyl-4-hydroxyphenyl) 1,2-naphthoquinone,2-(3,5-di-tert-butyl-4-hydroxyphenyl) 3-bromo-1,4-naphthoquinone,2-(3,5-di-tert-butyl-4-hydroxyphenyl) 3-chloro-1,4-naphthoquinone,2-(3,5-di-tert-butyl-4-hydroxyphenyl) 3-methoxy-1,4-naphthoquinone,2-(3,5-di-tert-butyl-4-hydroxyphenyl) 3-hydroxy-1,4-naphthoquinone,2-(3,5-di-tert-butyl-4-hydroxyphenyl) 1,4-naphthoquinone,4-(3,5-di-tert-butyl-4-hydroxyphenyl)3-hydroxy-5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-1,2-anthraquinone,4-(3,5-di-tert-butyl-4-hydroxyphenyl)3-methoxy-5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-1,2-anthraquinone,4-(3,5-di-tert-butyl-4-hydroxyphenyl)-5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-1,2-anthraquinone,3-bromo-4-(3,5-di-tert-butyl-4-hydroxyphenyl)-5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-1,2-anthraquinone,2-(3,5-di-tert-butyl-4-oxocyclohexa-2,5-dienylidine) indan-1,3-dione,2-(3,5-di-tert-butyl-4-oxocyclohexa-2,5-dienylidine)3,4-epoxy-3-hydroxy-4-methoxy-3,4-dihydro-2H-naphthalen-1-one,2-(3,5-di-tert-butyl-4-oxocyclohexa-2,5-dienylidine)3,4-epoxy-3,4-dimethoxy-3,4-dihydro-2H-naphthalen-1-one,2-(3,5-di-tert-butyl-4-hydroxyphenyl) indan-1-one,3,3-bi-[2-(3,5-di-tert-butyl-4-hydroxyphenyl)indan-1-on]-3-yl,2-(3,5-di-tert-butyl-4-hydroxyphenyl)3-bromo-5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-1,4-anthraquinone,2-(3,5-di-tert-butyl-4-hydroxyphenyl)3-chloro-5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-1,4-anthraquinone,2-(3,5-di-tert-butyl-4-hydroxyphenyl)3-methoxy-5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-1,4-anthraquinone,2-(3,5-di-tert-butyl-4-hydroxyphenyl)3-hydroxy-5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-1,4-anthraquinone,2-(3,5-di-tert-butyl-4-hydroxyphenyl)-5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-1,4-anthraquinone,2-bromo-3-(3-bromo-5-tert-butyl-4-hydroxyphenyl)-5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-1,4-anthraquinone,2-bromo-3-(3,5-dibromo-4-hydroxyphenyl)-5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-1,4-anthraquinone,2-bromo-3-(3-bromo-5-tert-butyl-4-hydroxyphenyl)3-hydroxy-5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-1,4-anthraquinone,3-bromo-2-(3,5-di-tert-butyl-4-hydroxyphenyl) 1,4-anthroquinone,2-(3,5-di-tert-butyl-4-hydroxyphenyl) 3-methoxy-1,4-anthraquinone,2-(3,5-di-tert-butyl-4-hydroxyphenyl) 3-hydroxy-1,4-anthraquinone,5,5,8,8-tetramethyl-5,6,7,8-tetrahydronapthalene-1,3-diol,3-methoxy-5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-1-ol,4-(3-chloro-5,5,8,8-tetramethyl-1,4-dioxo-1,4,5,6,7,8-hexahydroanthracen-2-yl)benzoic acid,methyl-4-(3-chloro-5,5,8,8-tetramethyl-1,4-dioxo-1,4,5,6,7,8-hexahydroanthracen-2-yl)benzoate, 4-(3-hydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl) benzoicacid, methyl-(3-methoxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl) benzoicacid,4-(3-hydroxy-5,5,8,8-tetramethyl-1,4-dioxo-1,4,5,6,7,8-hexahydroanthracen-2-yl)benzoic acid,methyl-4-(3-hydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl-azo) benzoate,4-(3-hydroxy-5,5,8,8-tetramethyl-1,4-dioxo-1,4,5,6,7,8-hexahydroanthracen-2-yl-azo)benzoic acid, 3-(3,5-di-tert-butyl-4-oxocyclohexa-2,5-dienylidine)5,5,8,8-tetramethyl-5,6,7,8-tetrahydrocyclopenta[b]naphthalen-1,2-dione,3-(3,5-di-tert-butyl-4-oxocyclohexa-2,5-dienylidene)5,5,8,8-tetramethyl-5,6,7,8-tetrahydroanthracen-3H-1,2,4-trione,2-(3,5-di-tert-butyl-4-hydroxyphenyl)3-methoxy-5,8-dimethyl-1,4-naphthoquinone,2-(3,5-di-tert-butyl-4-hydroxyphenyl)3-methoxy-6,7-dimethyl-1,4-naphthoquinone,2-(3,5-di-tert-butyl-4-hydroxyphenyl)3-methoxy-5-methyl-1,4-naphthoquinone,2-(3,5-di-tert-butyl-4-hydroxyphenyl)2-methoxy-5-methyl-1,4-naphthoquinone,2-(3,5-di-tert-butyl-4-hydroxyphenyl)3-methoxy-6-methyl-1,4-naphthoquinone,3-(3,5-di-tert-butyl-4-hydroxyphenyl)2-methoxy-6-methyl-1,4-naphthoquinione,2-(3,5-di-tert-butyl-4-hydroxyphenyl)3-methoxy-5,6-dimethyl-1,4-naphthoquinione,3-(3,5-di-tert-butyl-4-hydroxyphenyl)2-methoxy-5,6-dimethyl-1,4-naphthoquinone,2-(3,5-di-tert-butyl-4-hydroxyphenyl)-3-methoxy-5,7-dimethyl-1,4-naphthoquinone,3-(3,5-di-tert-butyl-4-hydroxyphenyl)2-methoxy-5,7-dimethyl-1,4-naphthoquinone,2-(3,5-di-tert-butyl-4-hydroxyphenyl)3-ethylthio-5-methyl-1,4-naphthoquinone,2-(3,5-di-tert-butyl-4-hydroxyphenyl)3-ethylthio-6-methyl-1,4-naphthoquinone,2-(3,5-di-tert-butyl-4-hydroxyphenyl)-3-hydroxy-5,8-dimethyl-1,4-naphthoquinone,2-(3,5-di-tert-butyl-4-hydroxyphenyl)-3-hydroxy-6,7-dimethyl-1,4-naphthoquinone,2-(3,5-di-tert-butyl-4-hydroxyphenyl)3-hydroxy-5-methyl-1,4-naphthoquinone,3-(3,5-di-tert-butyl-4-hydroxyphenyl)-2-hydroxy-5-methyl-1,4-naphthoquinone,2-(3,5-di-tert-butyl-4-hydroxyphenyl)-3-hydroxy-6-methyl-1,4-naphthoquinone,3-(3,5-di-tert-butyl-4-hydroxyphenyl)-2-hydroxy-6-methyl-1,4-naphthoquinone,2-(3,5-di-tert-butyl-4-hydroxyphenyl)3-hydroxy-5,6-dimethyl-1,4-naphthoquinone,2-(3-bromo-5-tert-butyl-4-hydroxyphenyl)3-hydroxy-5,6-dimethyl-1,4-naphthoquinone,3-(3,5-di-tert-butyl-4-hydroxyphenyl)-2-hydroxy-5,6-dimethyl-1,4-naphthoquinone,2-(3,5-di-tert-butyl-4-hydroxyphenyl)3-hydroxy-5,7-dimethyl-1,4-naphthoquinone,3-(3,5-di-tert-butyl-4-hydroxyphenyl)2-hydroxy-5,7-dimethyl-1,4-naphthoquinone.

According to the invention, it is preferred that the genomic DNA sampleis thermally denatured prior to the treatment.

According to the invention, it is particularly preferred that step c) isconducted in two sub-steps as follows:

a) a PCR preamplification with at least one pair of primers of differentsequence, which hybridize nonspecifically to a DNA sample pretreatedaccording to claim 1 and thus resulting in more than one amplifiedproduct in the PCR step;

b) a PCR amplification of the product formed in the preamplificationwith primers of different sequence, which are identical each time with asegment of the DNA sample [(+)-strand or (−)-strand] pretreatedaccording to claim 1 or, vice-versa, are complementary [to it], andwhich specifically hybridize to the DNA to be amplified.

It is also preferred according to the invention that the amplificationof several DNA segments is conducted in one reaction vessel.

It is also preferred according to the invention that a heat-stable DNApolymerase is used for the polymerase reaction.

It is particularly preferred according to invention that a desulfonationof the DNA is conducted prior to step c) of the method of the invention.

It is also preferred that the PCR products are hybridized to anoligonucleotide array for the detection of the pretreated DNA and thenthe following sub-steps are conducted:

a) the amplified genomic DNA is hybridized to at least oneoligonucleotide with the formation of a duplex, wherein said hybridizedoligonucleotides are directly adjacent by their 3′ end or at a distanceof up to 10 bases to the positions that are to be investigated relativeto their methylation in the genomic DNA sample;

b) the oligonucleotide with known sequence of n nucleotides is extendedby at least one nucleotide by means of a polymerase, whereby thenucleotide bears a detectable label and the extension depends on themethylation state of the respective cytosine in the genomic DNA sample.

It is preferred according to the invention that the PCR products arehybridized to an oligonucleotide array for the detection of thepretreated DNA and then the following sub-steps are conducted:

a) a set of oligonucleotides is hybridized to the amplified genomic DNAwith the formation of a duplex, whereby this set of oligonucleotides iscomprised of two different species, and whereby the hybridizedoligonucleotides of the first species are directly adjacent by their 3′end or at a distance of up to 10 bases to the positions that are to beinvestigated relative to their methylation in the genomic DNA sample,and whereby the second oligonucleotide of the second species hybridizesto a second region of the target molecule, so that the 5′ end of theoligonucleotide of the second species is separated by a gap of the sizeof a single nucleotide or up to 10 nucleotides from the 3′ end of thehybridized oligonucleotide of the first species at the site of saidselected position;

(b) the oligonucleotide of the first species with known sequence of nnucleotides is extended by means of a polymerase by at most the numberof nucleotides that lie between the 3′ end of the oligonucleotide of thefirst species and the 5′ end of the oligonucleotide of the secondspecies, whereby the extension depends on the methylation state of therespective cytosine in the genomic DNA sample;

(c) the oligonucleotides are incubated in the presence of a ligase,whereby the adjacent oligonucleotide of the first species, which isextended by the polymerase reaction, and the oligonucleotide of thesecond species are connected and in this way a ligation product isobtained, as long as, in the preceding step, an extension of theoligonucleotide of the first species has resulted such that the 3′ endwith the present 3′-hydroxy function of the extended oligonucleotide isnow directly adjacent to the 5′ end of the oligonucleotide of the secondspecies.

It is particularly preferred according to the invention that theoligonucleotides of the first species that are used and/or theoligonucleotides of the second species that are used either contain onlythe bases T, A and C or the bases T, A and G.

It is also preferred according to the invention that the PCR productsare hybridized to an oligonucleotide array for the detection of thepretreated DNA and then the following sub-steps are conducted:

(a) the amplified genomic DNA is hybridized to at least oneoligonucleotide with known sequence of n nucleotides with the formationof a duplex, wherein said hybridized oligonucleotides hybridize by their3′ end partially or completely to the positions which are to beinvestigated with respect to their methylation in the genomic DNAsample;

(b) the oligonucleotide is extended by at least one nucleotide by meansof a polymerase, as long as it has hybridized to the position to beinvestigated by its 3′ terminus beforehand without erroneous basepairing, whereby at least one nucleotide bears a detectable label andthe extension depends on the methylation state of the respectivecytosine in the genomic DNA sample.

It is also preferred according to the invention that the PCR productsand/or extension products and/or ligation products are provided with adetectable label for the detection. It is particularly preferred thatthe labels are fluorescent labels and/or that the labels areradionuclides. It is particularly preferred that the labels of thenucleotides are removable mass labels, which can be detected in a massspectrometer.

It is also particularly preferred that the PCR products and/or extensionproducts and/or ligation products are detected as a whole in the massspectrometer and are thus clearly characterized by their mass. It isalso preferred according to the invention that a fragment of the PCRproducts and/or extension products and/or ligation products is detectedeach time in the mass spectrometer.

The method according to the invention is preferably also characterizedby the fact that the fragment of the PCR product and/or extensionproduct and/or ligation product is produced by digestion with one ormore exo- or endonucleases.

It is additionally preferred that the produced fragments are providedwith an individual positive or negative net charge for betterdetectability in the mass spectrometer.

It is most particularly preferred that the PCR products and/or extensionproducts and/or ligation products are detected and visualized by meansof matrix-assisted laser desorption/ionization mass spectrometry(MALDI-TOF) or by means of electrospray mass spectrometry (ESI).

The method according to the invention is also preferred in which thegenomic DNA is obtained from a DNA sample, whereby sources for DNAinclude, e.g., cell lines, blood, sputum, stool, urine, cerebrospinalfluid, tissue embedded in paraffin, for example, tissue from eyes,intestine, kidneys, brain, heart, prostate, lungs, breast or liver,histological slide preparations and all possible combinations thereof.

Another subject of the present invention is the use of a methodaccording to the invention for the diagnosis and/or prognosis of adverseevents for patients or individuals, whereby these adverse events belongto at least one of the following categories: undesired druginteractions; cancer diseases; CNS malfunctions, damage or disease;symptoms of aggression or behavioral disturbances; clinical,psychological and social consequences of brain damage; psychoticdisturbances and personality disorders; dementia and/or associatedsyndromes; cardiovascular disease, malfunction and damage; malfunction,damage or disease of the gastrointestinal tract; malfunction, damage ordisease of the respiratory system; lesion, inflammation, infection,immunity and/or convalescence; malfunction, damage or disease of thebody as an abnormality in the development process; malfunction, damageor disease of the skin, of the muscles, of the connective tissue or ofthe bones; endocrine and metabolic malfunction, damage or disease;headaches or sexual malfunction.

The subject of the invention is also the use of a method according tothe invention for distinguishing cell types or tissues or forinvestigating cell differentiation.

Another subject of the present invention is finally a kit, comprised ofa reagent containing bisulfite, denaturing reagents or solvents, as wellas radical traps and primers for the production of amplified products,as well as instructions for conducting an assay according to a method ofthe invention.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a gel image of a PCR-amplified bisulfite-treated DNA strandobtained according to the Example.

DETAILED DESCRIPTION OF THE INVENTION

The present invention makes available a method for the detection ofmethylcytosine, which can be automated and which contains only pipettingsteps. The efficiency of existing methods is improved in this wayrelative to simplicity of manipulation, quality, costs, andparticularly, throughput.

A method that can be automated for the detection of methylcytosine ingenomic DNA samples is described:

The genomic DNA to be analyzed is preferably obtained from the usualsources for DNA, such as, e.g., cell lines, blood, sputum, stool, urine,cerebrospinal fluid, tissue embedded in paraffin, for example, tissuefrom eyes, intestine, kidneys, brain, heart, prostate, lungs, breast orliver, histological slide preparations and all possible combinationsthereof.

In the first step of the method, the DNA utilized is preferably treatedwith bisulfite (=disulfite, hydrogen sulfite) in such a way that allcytosines not methylated at the 5-position of the base are modified suchthat a base that differs relative to base pairing behavior is formed,while the cytosines that are methylated in the 5-position remainunchanged.

The genomic DNA sample is thermally denatured most preferably prior tothe treatment.

If bisulfite in the concentration range between 0.1 and 6 mol/l is usedfor the reaction, then an addition occurs at the unmethylated cytosinebases. For the method according to the invention, a denaturing reagentor solvent as well as a radical trap must also be present.

The following compounds or compound classes are considered preferably asdenaturing reagents or solvents:

Polyethylene glycol dialkyl ethers, dioxane and substituted derivatives,urea or derivatives, acetonitrile, primary alcohols, secondary alcohols,tertiary alcohols, diethylene glycol dialkyl ethers, triethylene glycoldialkyl ethers, tetraethylene glycol dialkyl ethers, pentaethylenedialkyl ethers, hexaethylene glycol dialkyl ethers, DMSO or THF.

The group of compounds listed in list 1 or their derivatives arepreferably suitable as radical traps. The subsequent alkaline hydrolysisthen leads to the conversion of unmethylated cytosine nucleobases touracil.

In the second step of the method, the treated DNA sample is diluted withwater or an aqueous solution. Then a desulfonation of the DNA ispreferably conducted (10-30 min, 90-100° C.) at alkaline pH.

In the third step of the method, the DNA sample is amplified in apolymerase chain reaction, preferably with a heat-stable DNA polymerase.The amplification of several DNA segments is preferably made in onereaction vessel.

The method step is preferably conducted in two sub-steps. One beginswith a PCR preamplification with at least one pair of primers ofdifferent sequence, which hybridize nonspecifically to the pretreatedDNA sample and thus produce more than one amplified product in the PCRstep. Then a PCR amplification of the product formed in thepreamplification is conducted with primers of different sequence, whichare identical each time to one segment of the pretreated DNA sample[(+)-strand or (−)-strand] or vice-versa, are complementary [to it] andspecifically hybridize to the DNA to be amplified.

It is thus clear that preamplifications of this type are frequentlyconducted not as PCR reactions, but as primer extension reactions, whichdo not require heat-stable polymerase.

In the last step of the method, one detects how much the sequence hasbeen changed due to the treatment with a reagent containing bisulfite incomparison to the genomic DNA sample and the methylation state of atleast one locus in the genomic DNA sample is concluded.

The PCR products are most preferably hybridized to an oligonucleotidearray for the detection.

In a preferred variant of the method, the following sub-steps areconducted after the hybridization to an oligonucleotide array:

a) the amplified genomic DNA is hybridized to at least oneoligonucleotide with the formation of a duplex, whereby said hybridizedoligonucleotides are directly adjacent by their 3′ end or at a distanceof up to 10 bases to the positions that are to be investigated relativeto their methylation in the genomic DNA sample;

(b) the oligonucleotide with known sequence of n nucleotides is extendedby at least one nucleotide by means of a polymerase, whereby thenucleotide bears a detectable label and the extension depends on themethylation state of the respective cytosine in the genomic DNA sample.

In another preferred variant of the method, the following sub-steps areconducted after the hybridization to an oligonucleotide array:

(a) a set of oligonucleotides is hybridized to the amplified genomic DNAwith the formation of a duplex, whereby this set of oligonucleotides iscomprised of two different species and whereby the hybridizedoligonucleotides of the first species are directly adjacent by their 3′end or at a distance of up to 10 bases to the positions that are to beinvestigated relative to their methylation in the genomic DNA sample,and whereby the second oligonucleotide of the second species hybridizesto a second region of the target molecule, so that the 5′ end of theoligonucleotide of the second species is separated by a gap of the sizeof one single nucleotide or up to 10 nucleotides from the 3′ end of thehybridized oligonucleotide of the first species at the site of saidselected position;

(b) the oligonucleotide of the first species with known sequence of nnucleotides is extended by means of a polymerase by at most the numberof nucleotides that lie between the 3′ end of the oligonucleotide of thefirst species and the 5′ end of the oligonucleotide of the secondspecies, whereby the extension depends on the methylation state of therespective cytosine in the genomic DNA sample;

(c) the oligonucleotides are incubated in the presence of a ligase,whereby the oligonucleotide of the first species that is extended by thepolymerase reaction and the oligonucleotide of the second species arejoined and a ligation product is obtained thereby, as long as anextension of the oligonucleotide of the first species has resulted inthe preceding step in such a way that now the 3′ end with the present 3′hydroxy function of the extended oligonucleotide is directly adjacent tothe 5′ end of the oligonucleotide of the second species, which ispreferably phosphorylated.

The oligonucleotides of the first species that is used and/or theoligonucleotides of the second species that is used contain in aparticularly preferred manner only the bases T, A and C or the bases T,A and G.

In another preferred variant of the method, the following sub-steps areconducted after the hybridization to an oligonucleotide array:

(a) the amplified genomic DNA is hybridized to at least oneoligonucleotide with known sequence of n nucleotides with the formationof a duplex, whereby said hybridized oligonucleotides hybridize by their3′ end either partially or completely to the positions that are to beinvestigated relative to their methylation in the genomic DNA sample;

(b) as long as it has hybridized by its 3′ terminus beforehand withouterroneous base pairing to the position to be investigated, theoligonucleotide is extended by at least one nucleotide by means of apolymerase, whereby at least one nucleotide bears a detectable label andthe extension depends on the methylation state of the respectivecytosine in the genomic DNA sample.

The PCR products and/or extension products and/or ligation products areparticularly preferably provided with a detectable label for thedetection.

Preferably, the labels of the PCR products and/or extension productsand/or ligation products are fluorescent labels, radionuclides orremovable mass labels, which are detected in a mass spectrometer.

The PCR products and/or extension products and/or ligation products canpreferably be detected as a whole in the mass spectrometer and are thusclearly characterized by their mass.

In a particularly preferred manner, a fragment of the PCR productsand/or extension products and/or ligation products is detected each timein the mass spectrometer.

The fragment of the PCR product and/or extension product and/or ligationproduct is preferably obtained by digestion with one or more exo- orendonucleases.

For better detectability in the mass spectrometer, the obtainedfragments most preferably have a single positive or negative net charge.

The PCR products and/or extension products and/or ligation products aredetected and visualized preferably by means of matrix assisted laserdesorption/ionization mass spectrometry (MALDI-TOF) or by means ofelectrospray mass spectrometry (ESI).

The present method is preferably used for the diagnosis and/or prognosisof adverse events for patients or individuals, whereby these adverseevents belong to at least one of the following categories: undesireddrug interactions; cancer diseases; CNS malfunctions, damage or disease;symptoms of aggression or behavioral disturbances; clinical,psychological and social consequences of brain damage; psychoticdisturbances and personality disorders; dementia and/or associatedsyndromes; cardiovascular disease, malfunction and damage; malfunction,damage or disease of the gastrointestinal tract; malfunction, damage ordisease of the respiratory system; lesion, inflammation, infection,immunity and/or convalescence; malfunction, damage or disease of thebody as an abnormality in the development process; malfunction, damageor disease of the skin, of the muscles, of the connective tissue or ofthe bones; endocrine and metabolic malfunction, damage or disease;headaches or sexual malfunction.

The new method also serves in a particularly preferred manner fordistinguishing cell types, tissues or for investigating celldifferentiation.

The subject of the present invention is also a kit, which contains areagent containing bisulfite, denaturing reagents or solvents, as wellas radical traps according to List 1, primers for the production of theamplified products and instructions for conducting an assay.

List 1:

Di- and trihyroxybenzenes, green tea extract, pine bark extract, gingkobiloba extract, (EGb 761), flavonoid mixture of different fruit andvegetable extracts (GMLD), Bio-Normalizer (Sun-O Corp), DPPH(1,1-diphenyl-2-picrylhydrazyl), NDGA (nondihydroguaiaretic acid),Trolox (6-hydroxy-2,5-7,8-tetramethylchromane 2-carboxylic acid),2,6-di-tert-butylphenol, 4-methyl-di-tert-butylphenol,4-methoxy-di-tert-butylphenol, 2,6-di-tert-butyl-p-cresol,3,4-dihydroxybenzoic acid, vitamin C, vitamin E, vitamin Q,hydroquinone, ubiquinone, lignans, hydroxyterpenes, flavonoids,curcumin, tannins, retinoic acid compounds, Ge-132bis-beta-carboxyethylgermanium sesquioxide, superoxide dismutase (SOD),superoxide catylase, alpha-naphthoflavone,di(2-methyl-5-chlorophenyl)dithionate and Cu(II) derivatives,mebendazole, CS (chloroform-soluble) alkyloid extract,4-(3,5-di-tert-butyl-4-hydroxyphenyl) 3-hydroxy-1,2-naphthoquinone,4-(3,5-di-tert-butyl-4-hydroxyphenyl) 3-methoxy-1,2-naphthoquinone,4-(3,5-di-tert-butyl-4-hydroxyphenyl) 1,2-naphthoquinone,2-(3,5-di-tert-butyl-4-hydroxyphenyl) 3-bromo-1,4-naphthoquinone,2-(3,5-di-tert-butyl-4-hydroxyphenyl) 3-chloro-1,4-naphthoquinone,2-(3,5-di-tert-butyl-4-hydroxyphenyl) 3-methoxy-1,4-naphthoquinone,2-(3,5-di-tert-butyl-4-hydroxyphenyl) 3-hydroxy-1,4-naphthoquinone,2-(3,5-di-tert-butyl-4-hydroxyphenyl) 1,4-naphthoquinone,4-(3,5-di-tert-butyl-4-hydroxyphenyl)3-hydroxy-5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-1,2-anthraquinone,4-(3,5-di-tert-butyl-4-hydroxyphenyl)3-methoxy-5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-1,2-anthraquinone,4-(3,5-di-tert-butyl-4-hydroxyphenyl)-5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-1,2-anthraquinone,3-bromo-4-(3,5-di-tert-butyl-4-hydroxyphenyl)-5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-1,2-anthraquinone,2-(3,5-di-tert-butyl-4-oxocyclohexa-2,5-dienylidine) indan-1,3-dione,2-(3,5-di-tert-butyl-4-oxocyclohexa-2,5-dienylidine)3,4-epoxy-3-hydroxy-4-methoxy-3,4-dihydro-2H-naphthalen-1-one,2-(3,5-di-tert-butyl-4-oxocyclohexa-2,5-dienylidine)3,4-epoxy-3,4-dimethoxy-3,4-dihydro-2H-naphthalen-1-one,2-(3,5-di-tert-butyl-4-hydroxyphenyl) indan-1-one,3,3-bi-[2-(3,5-di-tert-butyl-4-hydroxyphenyl)indan-1-on]-3-yl,2-(3,5-di-tert-butyl-4-hydroxyphenyl)3-bromo-5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-1,4-anthraquinone,2-(3,5-di-tert-butyl-4-hydroxyphenyl)3-chloro-5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-1,4-anthraquinone,2-(3,5-di-tert-butyl-4-hydroxyphenyl)3-methoxy-5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-1,4-anthraquinone,2-(3,5-di-tert-butyl-4-hydroxyphenyl)3-hydroxy-5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-1,4-anthraquinone,2-(3,5-di-tert-butyl-4-hydroxyphenyl)-5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-1,4-anthraquinone,2-bromo-3-(3-bromo-5-tert-butyl-4-hydroxyphenyl)-5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-1,4-anthraquinone,2-bromo-3-(3,5-dibromo-4-hydroxyphenyl)-5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-1,4-anthraquinone,2-bromo-3-(3-bromo-5-tert-butyl-4-hydroxyphenyl)3-hydroxy-5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-1,4-anthraquinone,3-bromo-2-(3,5-di-tert-butyl-4-hydroxyphenyl) 1,4-anthroquinone,2-(3,5-di-tert-butyl-4-hydroxyphenyl) 3-methoxy-1,4-anthraquinone,2-(3,5-di-tert-butyl-4-hydroxyphenyl) 3-hydroxy-1,4-anthraquinone,5,5,8,8-tetramethyl-5,6,7,8-tetrahydronapthalene-1,3-diol,3-methoxy-5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-1-ol,4-(3-chloro-5,5,8,8-tetramethyl-1,4-dioxo-1,4,5,6,7,8-hexahydroanthracen-2-yl)benzoic acid,methyl-4-(3-chloro-5,5,8,8-tetramethyl-1,4-dioxo-1,4,5,6,7,8-hexahydroanthracen-2-yl)benzoate, 4-(3-hydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl) benzoicacid, methyl-(3-methoxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl) benzoicacid,4-(3-hydroxy-5,5,8,8-tetramethyl-1,4-dioxo-1,4,5,6,7,8-hexahydroanthracen-2-yl)benzoic acid,methyl-4-(3-hydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl-azo) benzoate,4-(3-hydroxy-5,5,8,8-tetramethyl-1,4-dioxo-1,4,5,6,7,8-hexahydroanthracen-2-yl-azo)benzoic acid, 3-(3,5-di-tert-butyl-4-oxocyclohexa-2,5-dienylidine)5,5,8,8-tetramethyl-5,6,7,8-tetrahydrocyclopenta[b]naphthalen-1,2-dione,3-(3,5-di-tert-butyl-4-oxocyclohexa-2,5-dienylidene)5,5,8,8-tetramethyl-5,6,7,8-tetrahydroanthracen-3H-1,2,4-trione,2-(3,5-di-tert-butyl-4-hydroxyphenyl)3-methoxy-5,8-dimethyl-1,4-naphthoquinone,2-(3,5-di-tert-butyl-4-hydroxyphenyl)3-methoxy-6,7-dimethyl-1,4-naphthoquinone,2-(3,5-di-tert-butyl-4-hydroxyphenyl)3-methoxy-5-methyl-1,4-naphthoquinone,2-(3,5-di-tert-butyl-4-hydroxyphenyl)2-methoxy-5-methyl-1,4-naphthoquinone,2-(3,5-di-tert-butyl-4-hydroxyphenyl)3-methoxy-6-methyl-1,4-naphthoquinone,3-(3,5-di-tert-butyl-4-hydroxyphenyl)2-methoxy-6-methyl-1,4-naphthoquinione,2-(3,5-di-tert-butyl-4-hydroxyphenyl)3-methoxy-5,6-dimethyl-1,4-naphthoquinione,3-(3,5-di-tert-butyl-4-hydroxyphenyl)2-methoxy-5,6-dimethyl-1,4-naphthoquinone,2-(3,5-di-tert-butyl-4-hydroxyphenyl)-3-methoxy-5,7-dimethyl-1,4-naphthoquinone,3-(3,5-di-tert-butyl-4-hydroxyphenyl)2-methoxy-5,7-dimethyl-1,4-naphthoquinone,2-(3,5-di-tert-butyl-4-hydroxyphenyl)3-ethylthio-5-methyl-1,4-naphthoquinone,2-(3,5-di-tert-butyl-4-hydroxyphenyl)3-ethylthio-6-methyl-1,4-naphthoquinone,2-(3,5-di-tert-butyl-4-hydroxyphenyl)-3-hydroxy-5,8-dimethyl-1,4-naphthoquinone,2-(3,5-di-tert-butyl-4-hydroxyphenyl)-3-hydroxy-6,7-dimethyl-1,4-naphthoquinone,2-(3,5-di-tert-butyl-4-hydroxyphenyl)3-hydroxy-5-methyl-1,4-naphthoquinone,3-(3,5-di-tert-butyl-4-hydroxyphenyl)-2-hydroxy-5-methyl-1,4-naphthoquinone,2-(3,5-di-tert-butyl-4-hydroxyphenyl)-3-hydroxy-6-methyl-1,4-naphthoquinone,3-(3,5-di-tert-butyl-4-hydroxyphenyl)-2-hydroxy-6-methyl-1,4-naphthoquinone,2-(3,5-di-tert-butyl-4-hydroxyphenyl)3-hydroxy-5,6-dimethyl-1,4-naphthoquinone,2-(3-bromo-5-tert-butyl-4-hydroxyphenyl)3-hydroxy-5,6-dimethyl-1,4-naphthoquinone,3-(3,5-di-tert-butyl-4-hydroxyphenyl)-2-hydroxy-5,6-dimethyl-1,4-naphthoquinone,2-(3,5-di-tert-butyl-4-hydroxyphenyl)3-hydroxy-5,7-dimethyl-1,4-naphthoquinone,3-(3,5-di-tert-butyl-4-hydroxyphenyl)2-hydroxy-5,7-dimethyl-1,4-naphthoquinone.

The following example explains the invention.

Example Automated Conduction of the Bisulfite Reaction

The application of the method for detecting the methylation state ofcytosines in the factor VIII gene of a genomic DNA sample, which wastreated with a restriction endonuclease according to the instructions ofthe manufacturer, is described in the present example. The method isbased on the use of an automatic pipetting system (MWG RoboSeq 4204)with four separate vertically movable adapters for exchangeablepipetting tips, so as to exclude cross contaminations. The pipettingsystem makes possible the pipetting of 100 μl [aliquots] with an errorof less than ±2 μl. The operating plate of the automatic pipettingsystem is equipped with six racks for pipetting tips and eight pipettingpositions, two of which can be cooled, a reagent rack that can becooled, a stacking system for 10 microtiter plates, a pipette tipwashing station and a device for separating the pipette tips from theadapter.

The automatic pipetting system is connected to a computer by means of aserial interface and is controlled by means of a software program, whichpermits the free programming of all pipetting steps necessary for theapplication of the method.

In the first step of the method, an aliquot of the DNA sample ispipetted by hand into one of the 96 freely selectable positions of amicrotiter plate. The microtiter plate is then subsequently heated to96° C. with the use of an Eppendorf MasterCycler for denaturing thepretreated DNA sample. The microtiter plate is then transferred to theautomatic pipefting system. Aliquots of a denaturing agent (dioxane), a3.3 M sodium hydrogen sulfite solution, and a solution of a radical trapin the denaturing agent used are pipetted one after the other in aprogram-controlled manner from the reagent rack into all positions thatcontain DNA. Then the microtiter plate is incubated in the EppendorfMastercycler, so that all unmethylated cytosine residues in the DNAsample are converted into a bisulfite adduct with the action of thesodium hydrogen sulfite.

After the bisulfite treatment, the microtiter plate is transferred fromthe thermocycler to the automatic pipetting system. A second microtiterplate of the same type is then positioned. First, a basic Tris-HClbuffer (pH 9.5) and then an aliquot of the bisulfite-treated DNA aretransferred into the corresponding positions of the second microtiterplate in all chambers whose equivalent positions on the first microtiterplate contain a bisulfite-treated DNA sample. The bisulfite adducts ofthe unmethylated cytosine residues are converted to uracil residues inthe basic solution.

The targeted amplification of one strand (the sense strand in thepresent example) of the bisulfite-treated DNA is conducted by apolymerase chain reaction (PCR). A pair of primers of type 1 (AGG GAGTTT TTT TTA GGG AAT AGA GGG A (SEQ. ID NO: 1) and TAA TCC CAA AAC CTCTCC ACT ACA ACA A (SEQ ID NO: 2) are used, which permit the specificamplification of a successfully bisulfite-treated DNA strand, but not aDNA strand, whose unmethylated cytosine residues were not converted touracil residues or were incompletely converted. A third microtiter plateof the same type is positioned in the automatic pipetting system for thePCR reaction. In all chambers, whose equivalent positions on the firstmicrotiter plate contain a bisulfite-treated DNA sample, an aliquot of astock solution, which contains a PCR buffer, a DNA polymerase and aprimer of type 1 is first automatically pipetted. Then, an aliquot ofthe diluted bisulfite-treated DNA is transferred automatically from eachposition of the second microtiter plate to the corresponding position ofthe third microtiter plate, before the latter is transferred to thecycler for conducting the PCR reaction. The PCR product is identified byagarose gel electrophoresis and subsequent staining with ethidiumbromide (FIG. 1). FIG. 1 shows the gel image of a PCR-amplifiedbisulfite-treated DNA strand (left: molecular weight marker, right: PCRproduct).

1. A method for bisulfite treatment, wherein said method comprises: (a)providing a kit, said kit consisting of a reagent containing bisulfite,at least one denaturing solvent, at least one radical trap and primersfor production of amplified DNA; (b) incubating a genomic DNA samplewith a solution of the reagent containing bisulfite, wherein thebisulfite is in the concentration range between 0.1 and 6 mol/liter andin the presence of the at least one radical trap and the at least onedenaturing solvent; (c) diluting the treated DNA sample with water or anaqueous solution; (d) amplifying the DNA sample in a polymerase reactionusing the primers to yield amplified DNA; and (e) detecting how much thesequence has changed by the treatment according to step (b) incomparison to the genomic DNA sample and concluding the methylationstate of at least one locus in the genomic DNA sample.
 2. A method fordetecting the cytosine methylation of genomic DNA comprising, a)providing a kit consisting of a bisulfite solution in the concentrationrange between 0.1 and 6 mol/liter; at least one denaturing solvent, andat least one radical trap; b) incubating the genomic DNA sample with thesolution of bisulfite, the at least one denaturing solvent, and the atleast one radical trap; c) diluting the treated DNA sample with water oran aqueous solution; d) amplifying the DNA sample in a polymerasereaction in the presence of the primers to yield amplified DNA; e)detecting methylation changes in the DNA sequence in comparison to acontrol genomic DNA sample; and f) determining the methylation state ofat least one locus in the genomic DNA sample.
 3. The method according toclaim 2, wherein the radical trap is selected from the following groupof compounds: Di- and trihyroxybenzenes, green tea extract, pine barkextract, gingko biloba extract, DPPH (1,1-diphenyl-2-picrylhydrazyl),NDGA (nordihydroguaiaretic acid), 6-hydroxy-2,5-7,8-tetramethylchromane2-carboxylic acid, 2,6-di-tert-butylphenol,4-methyl-di-tert-butylphenol, 4-methoxy-di-tert-butylphenol,2,6-di-tert-butyl-p-cresol, 3,4-dihydroxybenzonic acid, vitamin C,vitamin E, vitamin Q, hydroquinone, ubiquinone, lignans,hydroxyterpenes, flavonoids, curcumin, tannins, retinoic acid compounds,Ge-132bis-beta-carboxyethylgermanium sesquioxide, superoxide dismutase(SOD), superoxide catylase, alpha-naphthoflavone,di(2-methyl-5-chlorophenyl)dithionate and Cu(II) derivatives,mebendazole, CS (chloroform-soluble) alkyloid extract,4-(3,5-di-tert-butyl-4-hydroxyphenyl) 3-hydroxy-1,2-naphthoquinone,4-(3,5-di-tert-butyl-4-hydroxyphenyl)-3-methoxy-1,2-naphthoquinone,4-(3,5-di-tert-butyl-4-hydroxyphenyl)1,2-naphthoquinone,2-(3,5-di-tert-butyl-4-hydroxyphenyl) 3-bromo-1,4-naphthoquinone,2-(3,5-di-tert-butyl-4-hydroxyphenyl) 3-chloro-1,4-naphthoquinone,2-(3,5-di-tert-butyl-4-hydroxyphenyl)-3-methoxy-1,4-naphthoquinone,2-(3,5-di-tert-butyl-4-hydroxyphenyl) 3-hydroxy-1,4-naphthoquinone,2-(3,5-di-tert-butyl-4-hydroxyphenyl)1,4-naphthoquinone,4-(3,5-di-tert-butyl-4-hydroxyphenyl)3-hydroxy-5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-1,2-anthraquinone,4-(3,5-di-tert-butyl-4-hydroxyphenyl)-3-methoxy-5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-1,2-anthraquinone,4-(3,5-di-tert-butyl-4-hydroxyphenyl)-5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-1,2-anthraquinone,3-bromo-4-(3,5-di-tert-butyl-4-hydroxyphenyl)-5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-1,2-anthraquinone,2-(3,5-di-tert-butyl-4-oxocyclohexa-2,5-dienylidine)indan-1,3-dione,2-(3,5-di-tert-butyl-4-oxocyclohexa-2,5-dienylidine)3,4-epoxy-3-hydroxy-4-methoxy-3,4-dihydro-2H-naphthalen-1-one,2-(3,5-di-tert-butyl-4-oxocyclohexa-2,5-dienylidine)3,4-epoxy-3,4-dimethoxy-3,4-dihydro-2H-naphthalen-1-one,2-(3,5-di-tert-butyl-4-hydroxyphenyl)indan-1-one,3,3-bi-[2-(3,5-di-tert-butyl-4-hydroxyphenyl)indan-1-on]-3-yl,2-(3,5-di-tert-butyl-4-hydroxyphenyl)3-bromo-5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-1,4-anthraquinone,2-(3,5-di-tert-butyl-4-hydroxyphenyl)3-chloro-5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-1,4-anthraquinone,2-(3,5-di-tert-butyl-4-hydroxyphenyl)-3-methoxy-5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-1,4-anthraquinone,2-(3,5-di-tert-butyl-4-hydroxyphenyl)3-hydroxy-5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-1,4-anthraquinone,2-(3,5-di-tert-butyl-4-hydroxyphenyl)-5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-1,4-anthraquinone,2-bromo-3-(3-bromo-5-tert-butyl-4-hydroxyphenyl)-5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-1,4-anthraquinone,2-bromo-3-(3,5-dibromo-4-hydroxyphenyl)-5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-1,4-anthraquinone,2-bromo-3-(3-bromo-5-tert-butyl-4-hydroxyphenyl)3-hydroxy-5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-1,4-anthraquinone,3-bromo-2-(3,5-di-tert-butyl-4-hydroxyphenyl)1,4-anthroquinone,2-(3,5-di-tert-butyl-4-hydroxyphenyl)-3-methoxy-1,4-anthraquinone,2-(3,5-di-tert-butyl-4-hydroxyphenyl)3-hydroxy-1,4-anthraquinone,5,5,8,8-tetramethyl-5,6,7,8-tetrahydronapthalen-1,3-diol,3-methoxy-5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-1-ol,4-(3-chloro-5,5,8,8-tetramethyl-1,4-dioxo-1,4,5,6,7,8-hexahydroanthracen-2-yl)benzoicacid,methyl-4-(3-chloro-5,5,8,8-tetramethyl-1,4-dioxo-1,4,5,6,7,8-hexahydroanthracen-2-yl)benzoate,4-(3-hydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)benzoic acid,methyl-(3-methoxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl benzoic acid,4-(3-hydroxy-5,5,8,8-tetramethyl-1,4-dioxo-1,4,5,6,7,8-hexahydroanthracen-2-yl)benzoicacid,methyl-4-(3-hydroxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl-azo)benzoate,4-(3-hydroxy-5,5,8,8-tetramethyl-1,4-dioxo-1,4,5,6,7,8-hexahydroanthracen-2-yl-azo)benzoicacid,3-(3,5-di-tert-butyl-4-oxocyclohexa-2,5-dienylidine)5,5,8,8-tetramethyl-5,6,7,8-tetrahydrocyclopenta[b]naphthalen-1,2-dione,3-(3,5-di-tert-butyl-4-oxocyclohexa-2,5-dienylidene)5,5,8,8-tetramethyl-5,6,7,8-tetrahydroanthracen-3H-1,2,4-trione,2-(3,5-di-tert-butyl-4-hydroxyphenyl)3-methoxy-5,8-dimethyl-1,4-naphthoquinone,2-(3,5-di-tert-butyl-4-hydroxyphenyl)3-methoxy-6,7-dimethyl-1,4-naphthoquinone,2-(3,5-di-tert-butyl-4-hydroxyphenyl)3-methoxy-5-methyl-1,4-naphthoquinone,2-(3,5-di-tert-butyl-4-hydroxyphenyl)2-methoxy-5-methyl-1,4-naphthoquinone,2-(3,5-di-tert-butyl-4-hydroxyphenyl)3-methoxy-6-methyl-1,4-naphthoquinone,3-(3,5-di-tert-butyl-4-hydroxyphenyl)2-methoxy-6-methyl-1,4-naphthoquinione,2-(3,5-di-tert-butyl-4-hydroxyphenyl)3-methoxy-5,6-dimethyl-4-naphthoquinione,3-(3,5-di-tert-butyl-4-hydroxyphenyl)2-methoxy-5,6-dimethyl-1,4-naphthoquinone,2-(3,5-di-tert-butyl-4-hydroxyphenyl)-3-methoxy-5,7-dimethyl-1,4-naphthoquinone,3-(3,5-di-tert-butyl-4-hydroxyphenyl)2-methoxy-5,7-dimethyl-1,4-naphthoquinone,2-(3,5-di-tert-butyl-4-hydroxyphenyl)3-ethylthio-5-methyl-1,4-naphthoquinone,2-(3,5-di-tert-butyl-4-hydroxyphenyl)3-ethylthio-6-methyl-1,4-naphthoquinone,2-(3,5-di-tert-butyl-4-hydroxyphenyl)-3-hydroxy-5,8-dimethyl-1,4-naphthoquinone,2-(3,5-di-tert-butyl-4-hydroxyphenyl)-3-hydroxy-6,7-dimethyl-1,4-naphthoquinone,2-(3,5-di-tert-butyl-4-hydroxyphenyl)-3-hydroxy-5-methyl-1,4-naphthoquinone,3-(3,5-di-tert-butyl-4-hydroxyphenyl)-2-hydroxy-5-methyl-1,4-naphthoquinone,2-(3,5-di-tert-butyl-4-hydroxyphenyl)-3-hydroxy-6-methyl-1,4-naphthoquinone,3-(3,5-di-tert-butyl-4-hydroxyphenyl)-2-hydroxy-6-methyl-1,4-naphthoquinone,2-(3,5-di-tert-butyl-4-hydroxyphenyl)3-hydroxy-5,6-dimethyl-1,4-naphthoquinone,2-(3-bromo-5-tert-butyl-4-hydroxyphenyl)3-hydroxy-5,6-dimethyl-1,4-naphthoquinone,3-(3,5-di-tert-butyl-4-hydroxyphenyl)-2-hydroxy-5,6-dimethyl-1,4-naphthoquinone,2-(3,5-di-tert-butyl-4-hydroxyphenyl)3-hydroxy-5,7-dimethyl-1,4-naphthoquinone,3-(3,5-di-tert-butyl-4-hydroxyphenyl)2-hydroxy-5,7-dimethyl-1,4-naphthoquinone.4. The method according to claim 2, wherein the genomic DNA has beenthermally denatured prior to use of the kit.
 5. The method according toclaim 2, wherein the primers comprise at least one pair of primers whichhybridize to the treated genomic DNA and at least one pair of primerswhich can hybridize to a DNA sample that has not been treated.
 6. Themethod according to claim 2, wherein the method is used for severalgenomic DNA segments.
 7. The method according to claim 2, wherein thegenomic DNA is obtained from cell lines, blood, sputum, stool, urine,cerebrospinal fluid, tissue embedded in paraffin, tissue of eyes,intestine, kidneys, brain, heart, prostate, lungs, breast or liver, orhistological slide preparations and all possible combinations thereof.8. The method according to claim 2, wherein the method is used for thediagnosis or prognosis of at least one category selected from the groupconsisting of: undesired drug interactions; cancer diseases; CNSmalfunctions, damage or disease; symptoms of aggression or behavioraldisturbances; clinical, psychological and social consequences of braindamage; psychotic disturbances and personality disorders; dementia;cardiovascular disease, malfunction and damage; malfunction, damage ordisease of the gastrointestinal tract; malfunction, damage or disease ofthe respiratory system; lesion, inflammation, infection, immunity andconvalescence; malfunction, damage or disease of the body as anabnormality in the development process; malfunction, damage or diseaseof the skin, of the muscles, of the connective tissue or of the bones;endocrine and metabolic malfunction, damage or disease; headaches orsexual malfunction.
 9. The method according to claim 2, wherein themethod is used for distinguishing cell types or tissues or forinvestigating cell differentiation.
 10. The method according to claim 2,comprising thermally denaturing the DNA prior to step b).
 11. The methodaccording to claim 2, comprising conducting a desulfonation of thetreated DNA for between 10 and 30 minutes at between 90° C. and 100° C.and at alkaline pH prior to step d).
 12. The method according to claim2, wherein the denaturing solvent is selected from the group consistingof: polyethylene glycol dialkyl ethers, dioxane and substitutedderivatives, urea derivatives, acetonitrile, secondary alcohols,tertiary alcohols, diethylene glycol dialkyl ethers, triethylene glycoldialkyl ethers, tetraethylene glycol dialkyl ethers, pentaethyleneglycol dialkyl ethers, hexaethylene glycol dialkyl ethers, and THF.